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Method For Rapidly Developing Gene Switches And Gene Circuits

Active Publication Date: 2013-10-10
CHIBA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a method for selecting a genetic switch and genetic circuit by using cells transfected with an expression vector harboring a genetic circuit and a gene sequence encoding a transcription regulatory factor whose expression is induced by a genetic switch. The cells are incubated in the presence of a compound that induces cell death under non-expression of the gene sequence controlled by the switch, and the viable cells are collected. This method allows for the rapid selection of genetic circuits with desired properties and functions, and is highly efficient and useful for synthetic biology.

Problems solved by technology

However, there are many proteins showing toxicity to the host cells.
However, the genetic switches included in the genetic circuit have problems of leakiness and cross-talk of switches, which correspond to leakage of electricity in an electronic circuit.
Hence, when a plurality of genetic switches are simultaneously used, the genetic switches do not properly work.
The de novo design of those requirements into a complex genetic circuit is an extremely difficult task.
Therefore, the limit of integration of a circuit in cell engineering is extremely low at present.
An “operon-type” selection method involving using an independent ON-selector and OFF-selector has a problem in that genetic mutations frequently occur in one of the selector genes, resulting in the frequent emergence of false positives.
Therefore, selection of a genetic circuit, in particular, selection of a complex genetic circuit, requires a large number of days.
As described above, the conventional approaches have the problem of requiring a long time period for a selection operation, or such problem that selection efficiency is affected by the selection conditions.

Method used

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  • Method For Rapidly Developing Gene Switches And Gene Circuits
  • Method For Rapidly Developing Gene Switches And Gene Circuits
  • Method For Rapidly Developing Gene Switches And Gene Circuits

Examples

Experimental program
Comparison scheme
Effect test

experimental example 1

[0172]An OFF selection method for a genetic switch and a genetic circuit was established.

[0173]First, with the use of mutation frequency in a low-concentration dP medium as an indicator, TK, thymidine monophosphate kinase (Tmk), and nucleotide diphosphate kinase (Ndk) were overexpressed. As a result, it was found that, as with other nucleoside analogs, the rate-limiting step of the incorporation of dP into DNA was the first phosphorylation catalyzed by TK (unpublished data). Next, some tk genes of different origins were tested. As a result, hsvTK showed the highest dP incorporation efficiency.

[0174]With the TK-deficient E. coli strain JW1226, human herpes virus derived thymidine kinase (hsvTK) was expressed by transfecting pTrc-hsvtk (FIG. 3), and a relationship between the concentration of dP and the survival rate of cells (viable cell count) was investigated.

[0175]First, JW1226 cells transfected with pTrc-hsvtk were cultured in 2 mL of LB medium containing 100 μg / mL of ampicillin ...

experimental example 2

[0180]An ON selection method for a genetic switch and a genetic circuit was established.

[0181]With the TK-deficient strain of E. coli JW1226, a human herpes virus derived thymidine kinase (hsvTK) was expressed by transfecting pTrc-hsvtk (FIG. 3), and a relationship between the concentration of 5FdU and the survival rate of cells (viable cell count) was investigated.

[0182]First, JW1226 cells transfected with pTrc-hsvtk were diluted with 1 mL of positive selection medium so as to achieve an OD600 of 0.002 (approximately 106 cells / mL), and cultured at 37° C. for 12 hours while being rotated at 200 rpm. The positive selection medium used was medium containing tryptone at 2% w / v, NaCl at 0.5% w / v, dT at 10 μg / mL, adenosine at 1 μg / mL, and 5FdU at 0 to 25 μg / mL. After the culture, a portion of the resultant culture was collected and inoculated in an LB plate containing ampicillin, and the number of colonies grown was counted as a viable cell count.

[0183]In the TK-deficient E. coli strain ...

experimental example 3

[0187]The rapidity of the OFF selection method established in Experimental Example 1 was studied.

[0188]First, TK-deficient cells transfected with pTrc-hsvtk (FIG. 3) so as to constantly express hsvTK were cultured under shaking at 37° C. and 200 rpm until an OD of 0.05 (approximately 3×107 cells / mL) was achieved. The resultant was dispensed at 1 mL each, dP was added (final concentration: 1 μM), and culture was continued at 37° C. After a lapse of 5, 30, or 60 minutes, 20 μL of the culture (approximately 106 cells) were collected and inoculated in an LB plate containing ampicillin, and the number of colonies grown was counted as a viable cell count.

[0189]In the sample to which a dP shock had been applied, the viable cell count was almost zero at any action time (FIG. 6). On the other hand, when the dP shock was not applied, the viable cell count increased. From the results, it was found that the OFF selection was sufficiently completed by an operation within up to 5 minutes.

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Abstract

Provided are: a selection method for a gene switch and a gene circuit, including using, as a selector, an expression vector containing at least a gene sequence whose expression is controlled by a transcription regulatory factor to be expressed when a genetic switch and a genetic circuit including the genetic switch operate, and a promoter sequence operably linked to the gene sequence upstream thereof; and an expression vector to be used in the selection method. This enables an effective selection method for a genetic switch and a genetic circuit, the selection method being able to be conducted within a short time period and with high selection efficiency and less leakiness.

Description

TECHNICAL FIELD[0001]The present invention relates to a developing method for a genetic switch and a genetic circuit. In more particular, the present invention relates to a developing method for a genetic switch and a genetic circuit, including selecting a genetic switch and a genetic circuit by using, as an indicator, cell death or cell death avoidance caused by expression or non-expression of a gene whose expression is controlled by an action of each of the genetic switch and the genetic circuit having the genetic switch. Specifically, the present invention relates to a selection method for a genetic switch and a genetic circuit, including using, as a selector, an expression vector containing at least: a gene sequence whose expression is controlled by a transcription regulatory factor to be expressed when a genetic switch and a genetic circuit including the genetic switch operate; and a promoter sequence operably linked to the gene sequence upstream thereof. The present invention ...

Claims

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Application Information

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IPC IPC(8): C12N15/70
CPCC12N15/63C12N15/635C12N15/70C12Q1/6897
Inventor UMENO, DAISUKEFUKUTOMI, HIROKISAITO, KYOICHI
Owner CHIBA UNIVERSITY
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